The present invention relates to a method of preparing a natural rubber latex and a method of preparing a deproteinized natural rubber latex, which are less likely to cause allergy, and a low allergenic natural rubber and a deproteinized natural rubber.
Natural rubbers have widely been used in various fields, for example, household appliances such as glove, medical appliances such as surgical glove and various catheters, lactation appliances, contraceptive device and the like because of features such as large extension, high elasticity and strong film strength. It has recently been reported that, when using medical appliances made of the natural rubber, such as surgical glove and various catheters, immediate (I type) allergy, which shows symptoms such as respiratory distress and anaphylactoid symptom (e.g. vascular edema, urttication, cyanosis, etc.), is caused. It is presumed that such immediate allergy is caused by a protein, as an antigen, in the natural rubber.
Accordingly, a trial of highly removing a protein in a natural rubber latex has recently been made and Japanese Patent No. 2,905,005 [Japanese Published Unexamined Patent (Kokai Tokkyo Koho Hei) No. 6-56902] discloses a method of adding a proteolytic enzyme such as alkali protease and a surfactant in a natural rubber latex, thereby subjecting to a deproteinization treatment, and sufficiently washing the latex by a centrifugation treatment.
According to the method described in the above patent publication, the protein in the natural rubber can be highly decomposed and removed. Specifically, the amount of the protein contained in the natural rubber, which is represented by the nitrogen content (N%) as determined by the Kjeldahl""s method, can be controlled to a very low value such as 0.02% or less. It is generally presumed that the natural rubber is a mixture of a high-molecular component having a number-average molecular weight  less than Mn greater than  of 1,000,000 to 2,500,000 and a low-molecular component having a number-average molecular weight of 100,000 to 200,000, the former high-molecular component being formed by mutually bonding the low-molecular components via peptide molecules contained in the natural rubber. Assuming that the molecular weight of the low-molecular component, which is deemed to be formed by original biosynthesis, is 100,000 and one peptide molecule in an intermolecular bond, i.e. one nitrogen atom (atomic weight: 14) is bonded based on one rubber molecule of this low-molecular component, the nitrogen content of the natural rubber is 0.014%. Accordingly, it is considered that about 0.02% of nitrogen is inevitably remained even if subjected to a high deproteinization treatment.
In case of subjecting to the deproteinization treatment by the method described in the above publication, an infrared absorption spectrum at 3280 cmxe2x88x921 peculiar to polypeptide is not observed in a rubber film formed by using the treated natural rubber latex. Therefore, it has been found that decomposition and removal of the protein are highly achieved by the method described in the above publication.
However, as is apparent from recent studies, there still exists a possibility of exhibiting allergy even if the protein is decomposed and removed through various conventionally known deproteinization treatments, including the method described in the above publication.
According to the present inventors,
(i) analysis was carried out by an analysis system of a protein, comprising a combination of a time of flight (TOF) type mass spectrometer and a two-dimensional surface decorative chip so as to confirm what degree a protein and a decomposition product thereof are remained in a natural rubber latex subjected to a deproteinization treatment by a conventional method, and
(ii) in vitro measurement based on an antigen-antibody reaction using serum of human patients was carried out in place of conventional in vivo measurement by a patch test so as to confirm allergy of a natural rubber latex subjected to a deproteinization treatment by a conventional method.
As a result, it has been found that:
(I) a protein or protein decomposition product having a number-average molecular weight  less than Mn greater than  of about 4500 to 4700 is remained and the protein is not sufficiently decomposed, and
(II) an allergenic protein is included in the amount enough to cause immediate allergy even after the deproteinization treatment.
The term xe2x80x9callergenic proteinxe2x80x9d is defined as follows in the present Invention. Any protein and decomposition product thereof (hereinafter referred to as a xe2x80x9cwhole proteinxe2x80x9d) in a sample of a natural rubber latex contain the group of an xe2x80x9cantigen proteinxe2x80x9d capable of producing an antibody in human serum. Antibodies produced in human serum are classified into an IgE-antibody capable of causing an allergic reaction and an antibody other than an IgE-antibody capable of causing no allergic reaction. Among the xe2x80x9cantigen proteinxe2x80x9d, the antigen protein capable of producing the IgE-antibody, which can cause the allergic reaction, is referred to as an xe2x80x9callergenic proteinxe2x80x9d so as to distinguish from other antigen proteins.
Thus, an object of the present invention is to provide a method of preparing a low allergenic natural rubber latex which substantially contains no allergenic protein and is therefore less likely to cause allergy.
Another object of the present invention is to provide a method of preparing a deproteinized natural rubber latex which substantially contains no allergenic protein and is therefore less likely to cause allergy.
Still another object of the present invention is to provide a natural rubber latex which is less likely to cause allergy because of high degree of the decomposition of a protein.
Further object of the present invention is to provide a deproteinized rubber latex which is less likely to cause allergy because of high degree of the decomposition and removal of a protein.
(I) The method of preparing a low allergenic natural rubber latex of the present invention has a feature that it comprises adding a protease having an exopeptidase activity to a natural rubber latex and aging the natural rubber latex, thereby to decompose a protein in the latex to such a degree that the protein and a protein decomposition product, which have a number-average molecular weight of 4500 or more, are not detected.
(II) The method of preparing a deproteinized natural rubber latex of the present invention has a feature that it comprises adding an alkali protease to a natural rubber latex, thereby to decompose a protein in the latex, adding a protease having an exopeptidase activity, thereby to further decompose the protein and a decomposition product thereof in the latex, and removing the protein and the decomposition product thereof.
(III) The low allergenic natural rubber of the present invention has a feature that it is a natural rubber obtained by a decomposition treatment of a protein, wherein the protein and a protein decomposition product, which have a number-average molecular weight of 4500 or more, are not detected.
(IV) The deproteinized natural rubber of the present invention has a feature that it is a natural rubber obtained by a decomposition treatment and a removing treatment of a protein, wherein the content of the protein is 0.02% or less in terms of a nitrogen content, an absorption at 3280 cmxe2x88x921 is not recognized in an infrared absorption spectrum, and the protein and a protein decomposition product, which have a number-average molecular weight of 4500 or more, are not detected.
A protease (proteolytic enzyme) used in a conventional deproteinization treatment is a protease produced mainly by bacteria, and the protease has an optimum pH in an alkali range and also has an endopeptidase activity but does not exhibit an exopeptidase activity. The reason why the alkali protease was mainly used in a conventional deproteinization treatment is as follows: (a) a field latex as a natural rubber is supplied in the form of a so-called ammonia latex prepared by concentrating the latex and adding ammonia for the purpose of preventing coagulation and putrefaction of the latex, and the latex itself exhibits alkalinity; (b) and, therefore, it was necessary to use a so-called alkali protease having an optimum pH in an alkali range so as to avoid coagulation of rubber molecules. Characteristics of a commercially available alkali protease, which has used in a conventional deproteinization treatment, are shown in Table 1.
The present inventors have made a trial of performing a decomposition treatment of a protein using a protease having an exopeptidase activity in place of a conventional alkali protease. As a result, they have surprisingly found a novel fact that the protein can be decomposed to such a degree that the protein and the protein decomposition product, which have a number-average molecular weight  less than Mn greater than  of 4500 or more, do not substantially exist, thereby making it possible to obtain a natural rubber latex which is less likely to cause allergy. Thus, the invention (I) of the above-described method of preparing a low allergenic natural rubber latex has been completed.
From another point of view, the present inventors have made a trial of performing a decomposition treatment of a protein using a protease having an exopeptidase activity and a removing treatment of the protein and the protein decomposition product thereof, in addition to the decomposition treatment of the protein using a conventional alkali protease. As a result, they have surprisingly found a novel fact that the protein can be decomposed and removed to such a degree that the protein and the protein decomposition product, which have a number-average molecular weight  less than Mn greater than  of 4500 or more do not substantially exist, thereby making it possible to obtain a deproteinized rubber latex which is less likely to cause allergy. Thus, the invention (II) of the above-described method of preparing a low allergenic natural rubber latex has been completed.
In the present invention, the protein and the protein decomposition product are usually detected by mass spectrometry, and preferably detected by using an analysis system of a protein, comprising a combination of a time of flight (TOF) type mass spectrometer and a two-dimensional surface decorative chip [for example, protein structure analyzer xe2x80x9cProtein Chip(trademark) Systemxe2x80x9d, manufactured by Ciphergen Biosystems, Inc.]. According to the Protein Chip(trademark) System, it is made possible to detect a protein in a trace amount such as about 0.1 xcexcg per 1 mL of a natural rubber latex.
Japanese Published Unexamined Patent (Kokai Tokkyo Koho Hei) No. 9-71604 describes that a surfactant is added to a natural rubber latex and, after adjusting the pH by adding a neutralizing agent, a proteolytic enzyme is added, thereby to decompose a protein in the latex. However, the publications merely describes about a decomposition treatment of a protein using an enzyme and does not disclose to what degree a molecular weight of the protein is reduced by such a treatment. The invention described in the publication has a feature in an extraction treatment, which comprises decomposing a protein using an enzyme, incorporating a vulcanizing agent in a latex, forming and vulcanizing the latex, dipping the vulcanized latex in a diluted alkali solution, and extracting the decomposed protein.
In the method of preparing a low allergenic natural rubber latex of the present invention (I), it is preferred to remove the protein and the decomposition product thereof in the latex after decomposing the protein in view of further reduction of allergy. Such removal treatment is preferably carried out by a centrifugation treatment in view of the removing effect and the efficiency of the removal treatment.
In the method of preparing a deproteinized natural rubber latex of the present invention (II), it is preferred to remove the protein and the decomposition product thereof by a centrifugation treatment in view of the removing effect and the efficiency of the removal treatment.
In the preparation methods of the present inventions (I) and (II), the pH of the latex is preferably adjusted to a neutral range (specifically pH 6 to 9) before treating with a protease having an exopeptidase. The reason is as follows. That is, since almost any protease having an exopeptidase activity has an optimum pH in the range of 6 to 9, and preferably about 6.5 to 8.5, the effect of the protein decomposition treatment can be enhanced if the pH of the latex is in the neutral range.
The protease having an exopeptidase activity used in the present inventions (I) and (II) is preferably a protease produced by microorganisms, which belong to the genus Aspergillus or Rhizopus, as a kind of filamentous bacteria. The peptidase derived from filamentous bacteria is suited for high degree decomposition of the protein.
The protease having an exopeptidase activity is more preferably a protease produced by microorganisms, which belong to Aspergillus oryzae, microorganisms which belong to Aspergillus mellus, or a protease produced by microorganisms which belong to Rhizopus oryzae, among those produced by microorganisms which belong to the genera described above.
In the preparation methods of the present inventions (I) and (II), the decomposition treatment of the protein is preferably carried out in the presence of a surfactant from such a view point that rubber molecules are stably dispersed in the latex, thereby to improve the decomposition of the protein.
Regarding the low allergenic natural rubber of the present invention (III), as is apparent from the fact that the protein and the protein decomposition product, which have a number-average molecular weight  less than Mn greater than  of 4500 or more, are not detected, the protein is highly decomposed as compared with the case when subjected to a deproteinization treatment by a conventionally known method. Therefore, it is considered that almost any portion, which can serve as an antigen capable of an IgE-antibody against human serum, is eliminated or denatured by the decomposition treatment, and allergy is less likely to occur.
Regarding the low allergenic natural rubber of the present invention (III), since the protein highly decomposes, a possibility of causing allergy is markedly reduced. Accordingly, it is suited for use as raw materials of household appliances such as gloves, medical appliances such as a surgical catheter, lactation appliances, contraceptive device and the like.
Regarding the deproteinized natural rubber of the present invention (IV), it is apparent that the total amount of the protein contained in the natural rubber is highly reduced similar to a conventional deproteinization treatment because the content of the protein is 0.02% or less in terms of a nitrogen content and an absorption at 3280 cmxe2x88x921 is not recognized in an infrared absorption spectrum.
Moreover, as is apparent from the fact that the protein and the protein decomposition product, which have a number-average molecular weight  less than Mn greater than  of 4500 or more, are not detected, the protein is highly decomposed as compared with the natural rubber subjected to a deproteinization treatment by a conventionally known method. Therefore, it is considered that almost any portion, which can serve as an antigen capable of an IgE-antibody against human serum, is eliminated or denatured by the decomposition treatment, and an allergic reaction is less likely to occur.
Regarding the deproteinized natural rubber of the present invention (IV), since the protein highly decomposes, a possibility of causing allergy is markedly reduced. Accordingly, it is suited for use as raw materials of household appliances such as glove, medical appliances such as a surgical catheter, lactation appliances, contraceptive device and the like.